Substituted pyrazolo[1,5-A]pyrimidines as tyrosine kinase inhibitors

ABSTRACT

The present invention relates to compounds which inhibit, regulate and/or modulate tyrosine kinase signal transduction, compositions which contain these compounds, and methods of using them to treat tyrosine kinase-dependent diseases and conditions, such as angiogenesis, cancer, tumor growth, atherosclerosis, age related macular degeneration, diabetic retinopathy, macular edema, retinal ischemia, inflammatory diseases, and the like in mammals.

PRIORITY CLAIM

This application is a §371 application of PCT/US03/40139 that was filedon Dec. 5, 2003,which claims priority from the U.S. ProvisionalApplication Nos. 60/432,445, filed on Dec. 11, 2002, now expired.

BACKGROUND OF THE INVENTION

The present invention relates to compounds which inhibit, regulateand/or modulate tyrosine kinase signal transduction, compositions whichcontain these compounds, and methods of using them to treat tyrosinekinase-dependent diseases and conditions, such as angiogenesis, cancer,tumor growth, atherosclerosis, age related macular degeneration,diabetic retinopathy, inflammatory diseases, and the like in mammals.

The following is provided as background information only and should notbe taken as an admission that any subject matter discussed or that anyreference mentioned is prior art to the instant invention.

Tyrosine kinases are a class of enzymes that catalyze the transfer ofthe terminal phosphate of adenosine triphosphate to tyrosine residues inprotein substrates. Tyrosine kinases are believed, by way of substratephosphorylation, to play critical roles in signal transduction for anumber of cell functions. Though the exact mechanism of signaltransduction is still unclear, tyrosine kinases have been shown to beimportant contributing factors in cell proliferation, carcinogenesis andcell differentiation,

Tyrosine kinases can be categorized as receptor type or non-receptortype. Receptor type tyrosine kinases have an extracellular, atransmembrane, and an intracellular portion, while non-receptor typetyrosine kinases are wholly intracellular.

The receptor-type tyrosine kinases are comprised of a large number oftransmembrane receptors with diverse biological activity. In fact, abouttwenty different subfamilies of receptor-type tyrosine kinases have beenidentified. One tyrosine kinase subfamily, designated the HER subfamily,is comprised of EGFR, HER2, HER3, and HER4. Ligands of this subfamily ofreceptors include epithileal growth factor, TGF-α, amphiregulin, HB-EGF,betacellulin and heregulin. Another subfamily of these receptor-typetyrosine kinases is the insulin subfamily, which includes INS-R, IGF-IR,and IR-R. The PDGF subfamily includes the PDGF-α and β receptors, CSFIR,c-kit and FLK-II. Then there is the FLK family which is comprised of thekinase insert domain receptor (KDR), fetal liver kinase-1 (FLK-1), fetalliver kinase-4 (FLK-4) and the fms-like tyrosine kinase-1 (flt-1). ThePDGF and FLK families are usually considered together due to thesimilarities of the two groups. For a detailed discussion of thereceptor-type tyrosine kinases, see Plowman et al., DN&P 7(6):334-339,1994, which is hereby incorporated by reference.

The non-receptor type of tyrosine kinases is also comprised of numeroussubfamilies, including Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak,Jak, Ack, and LIMK. Each of these subfamilies is further sub-dividedinto varying receptors. For example, the Src subfamily is one of thelargest and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, and Yrk.The Src subfamily of enzymes has been linked to oncogenesis. For a moredetailed discussion of the non-receptor type of tyrosine kinases, seeBolen Oncogene, 8:2025-2031 (1993), which is hereby incorporated byreference.

Both receptor-type and non-receptor type tyrosine kinases are implicatedin cellular signaling pathways leading to numerous pathogenicconditions, including cancer, psoriasis and hyperimmune responses.

Several receptor-type tyrosine kinases, and the growth factors that bindthereto, have been suggested to play a role in angiogenesis, althoughsome may promote angiogenesis indirectly (Mustonen and Alitalo, J. CellBiol. 129:895-898, 1995). One such receptor-type tyrosine kinase isfetal liver kinase 1 or FLK-1. The human analog of FLK-1 is the kinaseinsert domain-containing receptor KDR, which is also known as vascularendothelial cell growth factor receptor 2 or VEGFR-2, since it bindsVEGF with high affinity. Finally, the murine version of this receptorhas also been called NYK (Oelrichs et al., Oncogene 8(1):11-15, 1993).VEGF and KDR are a ligand-receptor pair that play an important role inthe proliferation of vascular endothelial cells, and the formation andsprouting of blood vessels, termed vasculogenesis and angiogenesis,respectively.

Angiogenesis is characterized by excessive activity of vascularendothelial growth factor (VEGF). VEGF is actually comprised of a familyof ligands (Klagsburn and D'Amore, Cytokine & Growth Factor Reviews7:259-270, 1996). VEGF binds the high affinity membrane-spanningtyrosine kinase receptor KDR and the related fms-like tyrosine kinase-1,also known as Flt-1 or vascular endothelial cell growth factor receptor1 (VEGFR-1). Cell culture and gene knockout experiments indicate thateach receptor contributes to different aspects of angiogenesis. KDRmediates the mitogenic function of VEGF whereas Flt-1 appears tomodulate non-mitogenic functions such as those associated with cellularadhesion. Inhibiting KDR thus modulates the level of mitogenic VEGFactivity. In fact, tumor growth has been shown to be susceptible to theantiangiogenic effects of VEGF receptor antagonists. (Kim et al., Nature362, pp. 841-844, 1993).

Solid tumors can therefore be treated by tyrosine kinase inhibitorssince these tumors depend on angiogenesis for the formation of the bloodvessels necessary to support their growth. These solid tumors includehistiocytic lymphoma, cancers of the brain, genitourinary tract,lymphatic system, stomach, larynx and lung, including lungadenocarcinoma and small cell lung cancer. Additional examples includecancers in which overexpression or activation of Raf-activatingoncogenes (e.g., K-ras, erb-B) is observed. Such cancers includepancreatic and breast carcinoma. Accordingly, inhibitors of thesetyrosine kinases are useful for the prevention and treatment ofproliferative diseases dependent on these enzymes.

The angiogenic activity of VEGF is not limited to tumors. VEGF accountsfor most of the angiogenic activity produced in or near the retina indiabetic retinopathy. This vascular growth in the retina leads to visualdegeneration culminating in blindness. Ocular VEGF mRNA and protein areelevated by conditions such as retinal vein occlusion in primates anddecreased pO₂ levels in mice that lead to neovascularization.Intraocular injections of anti-VEGF monoclonal antibodies or VEGFreceptor immunofusions inhibit ocular neovascularization in both primateand rodent models. Regardless of the cause of induction of VEGF in humandiabetic retinopathy, inhibition of ocular VEGF is useful in treatingthe disease.

Expression of VEGF is also significantly increased in hypoxic regions ofanimal and human tumors adjacent to areas of necrosis. VEGF is alsoupregulated by the expression of the oncogenes ras, raf, src and mutantp53 (all of which are relevant to targeting cancer). Monoclonalanti-VEGF antibodies inhibit the growth of human tumors in nude mice.Although these same tumor cells continue to express VEGF in culture, theantibodies do not diminish their mitotic rate. Thus tumor-derived VEGFdoes not function as an autocrine mitogenic factor. Therefore, VEGFcontributes to tumor growth in vivo by promoting angiogenesis throughits paracrine vascular endothelial cell chemotactic and mitogenicactivities. These monoclonal antibodies also inhibit the growth oftypically less well-vascularized human colon cancers in athymic mice anddecrease the number of tumors arising from inoculated cells.

Viral expression of a VEGF-binding construct of Flk-1, Flt-1, the mouseKDR receptor homologue, truncated to eliminate the cytoplasmic tyrosinekinase domains but retaining a membrane anchor, virtually abolishes thegrowth of a transplantable glioblastoma in mice presumably by thedominant negative mechanism of heterodimer formation with membranespanning endothelial cell VEGF receptors. Embryonic stem cells, whichnormally grow as solid tumors in nude mice, do not produce detectabletumors if both VEGF alleles are knocked out. Taken together, these dataindicate the role of VEGF in the growth of solid tumors. Inhibition ofKDR or Flt-1 is implicated in pathological angiogenesis, and thesereceptors are useful in the treatment of diseases in which angiogenesisis part of the overall pathology, e.g., inflammation, diabetic retinalvascularization, as well as various forms of cancer since tumor growthis known to be dependent on angiogenesis. (Weidner et al., N. Engl. J.Med., 324, pp. 1-8, 1991).

Accordingly, the identification of small compounds which specificallyinhibit, regulate and/or modulate the signal transduction of tyrosinekinases is desirable and is an object of this invention.

SUMMARY OF THE INVENTION

The present invention relates to compounds that are capable ofinhibiting, modulating and/or regulating signal transduction of bothreceptor-type and non-receptor type tyrosine kinases. One embodiment ofthe present invention is illustrated by a compound of Formula I, and thepharmaceutically acceptable salts and stereoisomers thereof.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are useful in the inhibition of kinasesand are illustrated by a compound of Formula I:

wherein

-   a and b are independently 0 or 1;-   m is independently 0, 1 or 2;-   R¹ is:    -   1) C₂-C₆ alkenyl,    -   2) C₂-C₆ alkynyl,    -   3) C₁-C₈ alkyl,    -   4) halo    -   5) CN,    -   6) (C═O)NR^(a)R^(b),    -   7) (C═O)R^(c),    -   8) (C═O)OR^(c),    -   9) heterocyclyl, said heterocyclyl is substitued with at least        one substituent selected from:        -   a) C₀-C₆ alkyl-(C═O)NR^(a)R^(b),        -   b) C₀-C₆ alkyl-SO_(m)R^(d),        -   c) C₀-C₆ alkyl-CO₂R^(c),        -   d) C₁-C₆ alkyl-OR^(c),        -   e) C₁-C₆ alkyl-NR^(a)R^(b), or        -   f) C₀-C₆ alkyl-(C═O)-C₀-C₆ alkyl-OR^(c);-   R² is:    -   1) H,    -   2) C₁-C₈ alkyl,    -   3) C₀-C₆ alkyl-C≡C—R^(a),    -   4) C₀-C₆ alkyl-CR^(a)═C(R^(a))₂,    -   5) C₀-C₆ alkyl-C₁-C₃-cycloalkenyl,    -   6) C₁-C₆ alkyl-aryl,    -   7) COR^(c),    -   8) CO₂R^(c),    -   9) C₀-C₆ alkyl-N(R^(a))₂,    -   10) heterocyclyl,    -   11) halo,    -   12) N(R^(a))₂,    -   13) OR^(c),    -   14) NO₂, or    -   15) S(O)_(m)R^(d),    -   Said alkyl, heterocyclyl and cycloalkenyl is optionally        substitued with at least one substituent selected from R^(b),-   R³, R⁴ and R⁵ are independently selected from:    -   1) H, provided R³, R⁴ and R⁵ are not all H at the same time,    -   2) (C═O)_(a)O_(b)C₁-C₁₀ alkyl,    -   3) halo,    -   4) aryl,    -   5) heterocyclyl,    -   6) NO₂,    -   7) OR^(c),    -   8) (C═O)_(a)O_(b)C₁-C₆ alkyl-N(R^(a))₂,    -   9) (C═O)_(a)N(R^(a))₂,    -   10) S(O)_(m)—C₁-C₆ alkyl-N(R^(a))₂, and    -   11) C₁-C₆ alkyl-(C═O)N(R^(a))₂,    -   Said alkyl, aryl and heterocyclyl are optionally substituted        with at least one substituent selected from R^(b);-   R^(a) and R^(b) independently are selected from:    -   1) H,    -   2) C₁-C₆ alkyl,    -   3) C₂-C₆ alkenyl,    -   4) C₂-C₆ alkynyl,    -   5) C₃-C₁₀ cycloalkyl,    -   6) aryl,    -   7) heterocyclyl,    -   8) C₀-C₆ alkyl-(C═O)NR^(a)R^(b),    -   9) C₀-C₆ alkyl-SO_(m)R^(d),    -   10) C₀-C₆ alkyl-CO₂R^(c),    -   11) C₀-C₆ alkyl-OR^(c),    -   12) C₀-C₆ alkyl-NR^(a)R^(b), and    -   13) C₀-C₆ alkyl-(C═O)—C₀-C₆ alkyl-OR^(c),    -   Said alkyl, aryl and heterocyclyl are optionally substituted        with at least one substituent selected from R^(d);-   R^(c) independently is:    -   1) H,    -   2) Unsubstituted or substituted C₁-C₆ alkyl,    -   3) Unsubstituted or substituted C₂-C₆ alkenyl,    -   4) Unsubstituted or substituted C₂-C₆ alkynyl,    -   5) Unsubstituted or substituted C₃-C₁₀ cycloalkyl,    -   6) Unsubstituted or substituted aryl, or    -   7) Unsubstituted or substituted heterocyclyl;-   R^(d) independently is:    -   1) Unsubstituted or substituted C₁-C₆ alkyl,    -   2) Unsubstituted or substituted C₂-C₆ alkenyl,    -   3) Unsubstituted or substituted C₂-C₆ alkynyl,    -   4) Unsubstituted or substituted C₃-C₁₀ cycloalkyl,    -   5) Unsubstituted or substituted aryl, or    -   6) Unsubstituted or substituted heterocyclyl;        or a pharmaceutically acceptable salt or stereoisomer thereof.

A second embodiment of the instant invention is a compound asillustrated above by Formula I wherein:

-   R² is:    -   1) H,    -   2) C₁-C₈ alkyl,    -   3) C₀-C₆ alkyl-C≡C—R^(a),    -   4) C₀-C₆ alkyl-CR^(a)═C(R^(a))₂,    -   5) C₀-C₆ alkyl-C₁-C₃-cycloalkenyl,    -   6) COR^(c),    -   7) CO₂R^(c),    -   8) C₀-C₆ alkyl-N(R^(a))₂,    -   9) halo, or    -   10) OR^(c);    -   Said alkyl and cycloalkenyl is optionally substitued with at        least one substituent selected from R^(b),-   R³ and R⁵ are independently selected from:    -   1) H,    -   2) (C═O)_(a)O_(b)C₁-C₁₀ alkyl,    -   3) halo,    -   4) NO₂,    -   5) OR^(c), and    -   6) C₁-C₆ alkyl-(C═O)N(R^(a))₂    -   Said alkyl is optionally substituted with at least one        substituent selected from R^(b);        and all other substituents and variables are as defined in the        first embodiment;        or a pharmaceutically acceptable salt or stereoisomer thereof.

A third embodiment of the instant invention is a compound as illustratedabove by Formula I wherein:

-   R⁴ is aryl, which is optionally substituted with at least one    substituent selected from R^(b);-   and all other substituents and variables are as defined in the    second embodiment;-   or a pharmaceutically acceptable salt or stereoisomer thereof.

A further embodiment of the third embodiment is a compound asillustrated above by Formula I wherein:

-   R² is:    -   1) H, or    -   2) C₁-C₈ alkyl;-   R³ and R⁵ are independently selected from:    -   1) H,    -   2) (C═O)_(a)O_(b)C₁-C₁₀ alkyl,    -   3) halo, and    -   4) OR^(c),    -   Said alkyl is optionally substituted with at least one        substituent selected from R^(b); and all other substituents and        variables are as defined in the third embodiment;        or a pharmaceutically acceptable salt or stereoisomer thereof.

Examples of compounds of the instant invention are

-   6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid    ethyl ester;-   6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid;-   6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid    amide;-   6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carbonitrile;-   ethyl    5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]nicotinate;-   5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-N-methylnicotinamide;-   N-ethyl-5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]nicotinamide;-   N-cyclopropyl-5-[6-(4-methoxyphenyl)    pyrazolo[1,5-a]pyrimidin-3-yl]nicotinamide;-   5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-N-propylnicotinamide;-   5-[6-(3-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-N-methylnicotinamide;-   N-ethyl-5-[6-(3-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]nicotinamide;-   5-[6-(3-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-N-propylnicotinamide;-   N-cyclopropyl-5-(6-pyridin-4-ylpyrazolo[1,5-a]pyrimidin-3-yl)nicotinamide;-   N-propyl-5-(6-pyridin-4-ylpyrazolo[1,5-a]pyrimidin-3-yl)nicotinamide;    or a pharmaceutically acceptable salt or stereoisomer thereof.

Further specific examples of a compound of Formula I are:

-   5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-N-methylnicotinamide

-   N-ethyl-5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]nicotinamide

-   N-cyclopropyl-5-(6-pyridin-4-ylpyrazolo[1,5-a]pyrimidin-3-yl)nicotinamide

or a pharmaceutically acceptable salt or stereoisomer thereof.

Also included within the scope of the present invention is apharmaceutical composition which is comprised of a compound of Formula Ias described above and a pharmaceutically acceptable carrier. Thepresent invention also encompasses a method of treating or preventingcancer in a mammal in need of such treatment which is comprised ofadministering to said mammal a therapeutically effective amount of acompound of Formula I Specific cancers for treatment are selected fromcancers of the brain, genitourinary tract, lymphatic system, stomach,larynx, and lung. Another set of specific forms of cancer arehistiocytic lymphoma, lung adenocarcinoma, small cell lung cancers,pancreatic cancer, gioblastomas and breast carcinoma.

Also included is a method of treating or preventing a disease in whichangiogenesis is implicated, which is comprised of administering to amammal in need of such treatment a therapeutically effective amount of acompound of Formula I. Such a disease in which angiogenesis isimplicated is ocular diseases such as retinal vascularization, diabeticretinopathy, age-related macular degeneration, and the like.

Also included within the scope of the present invention is a method oftreating or preventing inflammatory diseases which comprisesadministering to a mammal in need of such treatment a therapeuticallyeffective amount of a compound of Formula 1. Examples of suchinflammatory diseases are rheumatoid arthritis, psoriasis, contactdermatitis, delayed hypersensitivity reactions, and the like.

Also included is a method of treating or preventing a tyrosinekinase-dependent disease or condition in a mammal which comprisesadministering to a mammalian patient in need of such treatment atherapeutically effective amount of a compound of Formula I. Thetherapeutic amount varies according to the specific disease and isdiscernable to the skilled artisan without undue experimentation.

A method of treating or preventing retinal vascularization which iscomprised of administering to a mammal in need of such treatment atherapeutically effective amount of compound of Formula 1 is alsoencompassed by the present invention. Methods of treating or preventingocular diseases, such as diabetic retinopathy and age-related maculardegeneration, are also part of the invention. Also included within thescope of the present invention is a method of treating or preventinginflammatory diseases, such as rheumatoid arthritis, psoriasis, contactdermatitis and delayed hypersensitivity reactions, as well as treatmentor prevention of bone associated pathologies selected from osteosarcoma,osteoarthritis, and rickets.

The invention also contemplates the use of the instantly claimedcompounds in combination with a second compound selected from:

1) an estrogen receptor modulator,

2) an androgen receptor modulator,

3) retinoid receptor modulator,

4) a cytotoxic agent,

5) an antiproliferative agent,

6) a prenyl-protein transferase inhibitor,

7) an HMG-CoA reductase inhibitor,

8) an HIV protease inhibitor,

9) a reverse transcriptase inhibitor,

10) an angiogenesis inhibitor,

11) PPAR-γ agonists,

12) PPAR-δ agonists,

13) an inhibitor of inherent multidrug resistance,

14) an anti-emetic agent,

15) an agent useful in the treatment of anemia,

16) agent useful in the treatment of neutropenia, and

17) an immunologic-enhancing drug.

Preferred angiogenesis inhibitors are selected from the group consistingof a tyrosine kinase inhibitor, an inhibitor of epidermal-derived growthfactor, an inhibitor of fibroblast-derived growth factor, an inhibitorof platelet derived growth factor, an MMP (matrix metalloprotease)inhibitor, an integrin blocker, interferon-α, interleukin-12, pentosanpolysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, troponin-1, and an antibody to VEGF. Preferredestrogen receptor modulators are tamoxifen and raloxifene.

Also included in the scope of the claims is a method of treating cancerwhich comprises administering a therapeutically effective amount of acompound of Formula 1 in combination with radiation therapy and/or incombination with a compound selected from:

1) an estrogen receptor modulator,

2) an androgen receptor modulator,

3) retinoid receptor modulator,

4) a cytotoxic agent,

5) an antiproliferative agent,

6) a prenyl-protein transferase inhibitor,

7) an HMG-CoA reductase inhibitor,

8) an HIV protease inhibitor,

9) a reverse transcriptase inhibitor,

10) an angiogenesis inhibitor,

11) PPAR-γ agonists,

12) PPAR-δ agonists,

13) an inhibitor of inherent multidrug resistance,

14) an anti-emetic agent,

15) an agent useful in the treatment of anemia,

16) agent useful in the treatment of neutropenia, and

17) an immunologic-enhancing drug.

And yet another embodiment of the invention is a method of treatingcancer which comprises administering a therapeutically effective amountof a compound of Formula 1 in combination with paclitaxel ortrastuzumab.

Also within the scope of the invention is a method of reducing orpreventing tissue damage following a cerebral ischemic event whichcomprises administering a therapeutically effective amount of a compoundof Formula I.

These and other aspects of the invention will be apparent from theteachings contained herein.

“Tyrosine kinase-dependent diseases or conditions” refers to pathologicconditions that depend on the activity of one or more tyrosine kinases.Tyrosine kinases either directly or indirectly participate in the signaltransduction pathways of a variety of cellular activities includingproliferation, adhesion and migration, and differentiation. Diseasesassociated with tyrosine kinase activities include the proliferation oftumor cells, the pathologic neovascularization that supports solid tumorgrowth, ocular neovascularization (diabetic retinopathy, age-relatedmacular degeneration, and the like) and inflammation (psoriasis,rheumatoid arthritis, and the like).

The compounds of the present invention may have asymmetric centers,chiral axes, and chiral planes (as described in E. L. Eliel and S. H.Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York,1994, pages 1119-1190), and occur as racemates, racemic mixtures, and asindividual diastereomers, with all possible isomers and mixturesthereof, including optical isomers, being included in the presentinvention. In addition, the compounds disclosed herein may exist astautomers and both tautomeric forms are intended to be encompassed bythe scope of the invention, even though only one tautomeric structure isdepicted. For example, any claim to compound A below is understood toinclude tautomeric structure B, and vice versa, as well as mixturesthereof.

When any substituent or variable (e.g. R^(a), R^(b), etc.) occurs morethan one time in any constituent, its definition on each occurrence isindependent at every other occurrence. Also, combinations ofsubstituents and variables are permissible only if such combinationsresult in stable compounds. Lines drawn into the ring systems fromsubstituents indicate that the indicated bond may be attached to any ofthe substitutable ring atoms. If the ring system is polycyclic, it isintended that the bond be attached to any of the suitable carbon atomson the proximal ring only.

It is intended that the definition of any substituent or variable (e.g.,R¹, R^(a), n, etc.) at a particular location in a molecule beindependent of its definitions elsewhere in that molecule. Thus,—N(R^(a))₂ represents —NHH, —NHCH₃, —NHC₂H₅, etc. It is understood thatsubstituents and substitution patterns on the compounds of the instantinvention can be selected by one of ordinary skill in the art to providecompounds that are chemically stable and that can be readily synthesizedby techniques known in the art, as well as those methods set forthbelow, from readily available starting materials.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

As used herein, “alkyl” is intended to include both branched andstraight-chain aliphatic hydrocarbon groups having the specified numberof carbon atoms. For example, C₁-C₁₀, as in “C₁-C₁₀ alkyl” is defined toinclude groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in alinear or branched arrangement. For example, “C₁-C₁₀ alkyl” specificallyincludes methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl,pentyl, hexyl, heptyl, and so on.

“Cycloalkyl” as used herein is intended to include non-aromatic cyclichydrocarbon groups, having the specified number of carbon atoms, whichmay or may not be bridged or structurally constrained. Examples of suchcycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, adamantyl, cyclooctyl, cycloheptyl,tetrahydro-naphthalene, methylenecylohexyl, and the like. As usedherein, examples of “C₃-C₁₀ cycloalkyl” may include, but are not limitedto:

“Alkoxy” represents an alkyl group of indicated number of carbon atomsas defined above attached through an oxygen bridge.

If no number of carbon atoms is specified, the term “alkenyl” refers toa non-aromatic hydrocarbon radical, which may be branched or unbranchedand cyclic or acyclic, containing from 2 to 10 carbon atoms and at leastone carbon to carbon double bond. Preferably one carbon to carbon doublebond is present, and up to four non-aromatic carbon-carbon double bondsmay be present. Thus, “C₂-C₆ alkenyl” means an alkenyl radical havingfrom 2 to 6 carbon atoms. Alkenyl groups include ethenyl, propenyl,butenyl, 2-methylbutenyl, cyclohexenyl, methylenylcyclohexenyl, and soon. As described above with respect to alkyl, the straight, branched orcyclic portion of the alkenyl group may contain double bonds and may besubstituted if a substituted alkenyl group is indicated.

The term “alkynyl” refers to a hydrocarbon radical, which may bebranched or unbranched and cyclic or acyclic, containing from 2 to 10carbon atoms and at least one carbon to carbon triple bond. Up to threecarbon-carbon triple bonds may be present. Thus, “C₂-C₆ alkynyl” meansan alkynyl radical having from 2 to 6 carbon atoms. Alkynyl groupsinclude ethynyl, propynyl, butynyl, 3-methylbutynyl and so on. Asdescribed above with respect to alkyl, the straight, branched or cyclicportion of the alkynyl group may contain triple bonds and may besubstituted if a substituted alkynyl group is indicated.

In certain instances, substituents may be defined with a range ofcarbons that includes zero, such as (C₀-C₆)alkyl-aryl. If aryl is takento be phenyl, this definition would include phenyl itself as well as—CH₂Ph, —CH₂CH₂Ph, CH(CH₃) CH₂CH(CH₃)Ph, and so on.

As used herein, “aryl” is intended to mean any stable monocyclic orbicyclic carbon ring of up to 7 atoms in each ring, wherein at least onering is aromatic. Examples of such aryl elements include phenyl,naphthyl, tetrahydronaphthyl, indanyl, indanonyl, indenyl, biphenyl,tetralinyl, tetralonyl, fluorenonyl, phenanthryl, anthryl, acenaphthyl,tetrahydronaphthyl, and the like. In cases where the aryl substituent isbicyclic, it is understood that attachment is via the phenyl ring.

As appreciated by those of skill in the art, “halo” or “halogen” as usedherein is intended to include chloro, fluoro, bromo and iodo.

The term heteroaryl, as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. Heteroaryl groups within the scope of thisdefinition include but are not limited to: acridinyl, carbazolyl,cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzodioxolyl,benzotriazolyl, benzothiofuranyl, benzothiazolyl, furanyl, thienyl,benzothienyl, benzofuranyl, benzoquinolinyl, isoquinolinyl, oxazolyl,isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl,pyrrolyl, quinolinyl, tetrahydronaphthyl, tetrahydroquinoline, and thelike.

The term heterocycle or heterocyclic or heterocyclyl, as used herein,represents a stable 5- to 7-membered monocyclic or stable 8- to11-membered bicyclic heterocyclic ring which is either saturated orunsaturated, and which consists of carbon atoms and from one to fourheteroatoms selected from the group consisting of N, O, and S, andincluding any bicyclic group in which any of the above-definedheterocyclic rings is fused to a benzene ring. The heterocyclic ring maybe attached at any heteroatom or carbon atom which results in thecreation of a stable structure. “Heterocycle” or “heterocyclyl”therefore includes the above mentioned heteroaryls, as well as dihydroand tetrathydro analogs thereof. Further examples of “heterocyclyl”include, but are not limited to the following: benzodioxolyl,benzofuranyl, benzofurazanyl, benzimidazolyl, benzopyranyl,benzopyrazolyl, benzotriazolyl, benzothiazolyl, benzothienyl,benzothiofuranyl, benzothiophenyl, benzothiopyranyl, benzoxazolyl,carbazolyl, carbolinyl, chromanyl, cinnolinyl, diazapinonyl,dihydrobenzodioxinyl, dihydrobenzofuranyl, dihydrobenzofuryl,dihydrobenzoimidazolyl, dihydrobenzothienyl, dihydrobenzothiopyranyl,dihydrobenzothiopyranyl sulfone, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrocyclopentapyridinyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxanyl,dioxidotetrahydrothienyl, furyl, furanyl, imidazolyl, imidazolinyl,imidazolidinyl, imidazothiazolyl, imidazopyridinyl, indazolyl,indolazinyl, indolinyl, indolyl, isobenzofuranyl, isochromanyl,isoindolyl, isoindolinyl, isoquinolinone, isoquinolyl, isothiazolyl,isothiazolidinyl, isoxazolinyl, isoxazolyl, methylenedioxybenzoyl,morpholinyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazolinyl,oxetanyl, oxoazepinyl, oxadiazolyl, oxodihydrophthalazinyl,oxodihydroindolyl, oxodihydrotriazolyl, oxoimidazolidinyl,oxopiperazinyl, oxopiperdinyl, oxopyrrolidinyl, oxopyrimidinyl,oxopyrrolyl, oxotriazolyl, piperidyl, piperidinyl, piperazinyl, pyranyl,pyrazinyl, pyrazolyl, pyridazinyl, pyridinonyl, pyridopyridinyl,pyridazinyl, pyridyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolidinyl,quinazolinyl, quinolinyl, quinolyl, quinolinonyl, quinoxalinyl,tetrahydrobenzoannulenyl, tetrahydrocycloheptapyridinyl,tetrahydrofuranyl, tetrahydrofuryl, tetrahydroisoquinolinyl,tetrahydropyranyl, tetrahydroquinolinyl, tetrazolyl, tetrazolopyridyl,thiadiazolyl, thiazolyl, thiazolinyl, thienofuryl, thienyl, triazolyl,azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, and the like. In oneembodiment of the instant invention, heterocycle is selected fromoxoazepinyl, benzimidazolyl, dioxanyl, dihydrobenzodioxinyl,dihydroindolyl, dihydrotriazolyl, dioxanyl, dioxidotetrahydrothienyl,oxetanyl, piperidinyl, pyrazolyl, pyridinyl, tetrahydrobenzoannulenyl,tetrahydrofuranyl, tetrahydropyranyl, tetrazolyl, imidazolyl, indolyl,isoquinolinyl, morpholinyl, piperidyl, piperazinyl, pyridyl,pyrrolidinyl, oxopiperidinyl, oxopyrrolidinyl, quinolinyl,tetrahydrofuryl, tetrahydroisoquinolinyl, thienyl, and triazolyl.

As used herein, “aralkyl” is intended to mean an aryl moiety, as definedabove, attached through a C₁-C₁₀ alkyl linker, where alkyl is definedabove. Examples of aralkyls include, but are not limited to, benzyl,naphthylmethyl and phenylpropyl.

As used herein, “heterocyclylalkyl” is intended to mean a heterocyclicmoiety, as defined below, attached through a C₁-C₁₀ alkyl linker, wherealkyl is defined above. Examples of heterocyclylalkyls include, but arenot limited to, pyridylmethyl, imidazolylethyl, pyrrolidinylmethyl,morpholinylethyl, quinolinylmethyl, imidazolylpropyl and the like.

As used herein, the terms “substituted C₁-C₁₀ alkyl” and “substitutedC₁-C₆ alkoxy” are intended to include the branch or straight-chain alkylgroup of the specified number of carbon atoms, wherein the carbon atomsmay be substituted with 1 to 3 substituents selected from the groupwhich includes, but is not limited to, halo, C₁-C₂₀ alkyl, CF₃, NH₂,N(C₁-C₆ alkyl)₂, NO₂, oxo, CN, N₃, —OH, —O(C₁-C₆ alkyl), C₃-C₁₀cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, (C₀-C₆ alkyl) S(O)₀₋₂—, (C₀-C₆alkyl)S(O)₀₋₂(C₀-C₆ alkyl)-, (C₀-C₆ alkyl)C(O)NH—, H₂N—C(N—, —O(C₁-C₆alkyl)CF₃, (C₀-C₆ alkyl)C(O)—, (C₀-C₆ alkyl)OC(O)—, (C₀-C₆ alkyl)O(C₁-C₆alkyl)-, (C₀-C₆ alkyl)C(O)₁₋₂(C₀-C₆ alkyl)-, (C₀-C₆ alkyl)OC(O)NH—,aryl, aralkyl, heterocycle, heterocyclylalkyl, halo-aryl, halo-aralkyl,halo-heterocycle, halo-heterocyclylalkyl, cyano-aryl, cyano-aralkyl,cyano-heterocycle and cyano-heterocyclylalkyl.

As used herein, the terms “substituted C₃-C₁₀ cycloalkyl”, “substitutedaryl”, “substituted heterocycle”, “substituted aralkyl” and “substitutedheterocyclylalkyl” are intended to include the cyclic group containingfrom 1 to 3 substituents in addition to the point of attachment to therest of the compound. Preferably, the substituents are selected from thegroup which includes, but is not limited to, halo, C₁-C₂₀ alkyl, CF₃,NH₂, N(C₁-C₆ alkyl)₂, NO₂, oxo, CN, N₃, —OH, —O(C₁-C₆ alkyl), C₃-C₁₀cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, (C₀-C₆ alkyl) S(O)₀₋₂—, (C₀-C₆alkyl)S(O)₀₋₂(C₀-C₆ alkyl)-, (C₀-C₆ alkyl)C(O)NH—, H₂N—C(NH)—, —O(C₁-C₆alkyl)CF₃, (C₀-C₆ alkyl)C(O)—, (C₀-C₆ alkyl)OC(O)—, (C₀-C₆alkyl)O(C₁-C₆alkyl)-, (C₀-C₆ alkyl)C(O)₁₋₂(C₀-C₆ alkyl)-, (C₀-C₆ alkyl) OC(O)NH—,aryl, aralkyl, heteroaryl, heterocyclylalkyl, halo-aryl, halo-aralkyl,halo-heterocycle, halo-heterocyclylalkyl, cyano-aryl, cyano-aralkyl,cyano-heterocycle and cyano-heterocyclylalkyl.

As used herein, the phrase “substituted with at least one substituent”is intended to mean that the substituted group being referenced has from1 to 6 substituents. In an aspect of the instant invention, thesubstituted group being referenced contains from 1 to 3 substituents, inaddition to the point of attachment to the rest of the compound.

In an aspect of the invention, R¹ is selected from halo, CN,(C═O)NR^(a)R^(b), (C═O)R^(c), (C═O)OR^(c), and heterocyclyl.

In an aspect of the invention, R² is selected from H and C₁-C₈ alkyl.

In another aspect of the invention, R³ and R⁵ are independently selectedfrom H and (C═O)_(a)O_(b)C₁-C₁₀ alkyl.

In an embodiment of the invention, R⁴ is an aryl. In a furtherembodiment of the instant invention, R⁴ is phenyl or naphthyl.

It is intended that the definition of any substituent or variable (e.g.,R¹, R^(1a), n, etc.) at a particular location in a molecule beindependent of its definitions elsewhere in that molecule. Thus,—N(R^(a))₂ represents —NHH, —NHCH₃, —NHC₂H₅, etc. It is understood thatsubstituents and substitution patterns on the compounds of the instantinvention can be selected by one of ordinary skill in the art to providecompounds that are chemically stable and that can be readily synthesizedby techniques known in the art, as well as those methods set forthbelow, from readily available starting materials.

For use in medicine, the salts of the compounds of Formula I will bepharmaceutically acceptable salts. Other salts may, however, be usefulin the preparation of the compounds according to the invention or oftheir pharmaceutically acceptable salts. When the compound of thepresent invention is acidic, suitable “pharmaceutically acceptablesalts” refers to salts prepared form pharmaceutically acceptablenon-toxic bases including inorganic bases and organic bases. Saltsderived from inorganic bases include aluminum, ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous,potassium, sodium, zinc and the like. Particularly preferred are theammonium, calcium, magnesium, potassium and sodium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as arginine, betaine caffeine, choline,N,N¹-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine tripropylamine, tromethamineand the like.

When the compound of the present invention is basic, salts may beprepared from pharmaceutically acceptable non-toxic acids, includinginorganic and organic acids. Such acids include acetic, benzenesulfonic,benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic,glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and thelike. Particularly preferred are citric, hydrobromic, hydrochloric,maleic, phosphoric, sulfuric and tartaric acids.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977:66:1-19.

It will also be noted that the compounds of the present invention arepotentially internal salts or zwitterions, since under physiologicalconditions a deprotonated acidic moiety in the compound, such as acarboxyl group, may be anionic, and this electronic charge might then bebalanced off internally against the cationic charge of a protonated oralkylated basic moiety, such as a quaternary nitrogen atom.

Abbreviations, which may be used in the description of the chemistry andin the Examples that follow, include:

-   Ac₂O Acetic anhydride;-   AcOH Acetic acid;-   AIBN 2,2′-Azobisisobutyronitrile;-   Ar Aryl;-   BINAP 2,2′-Bis(diphenylphosphino)-1,1′ binaphthyl;-   Bn Benzyl;-   BOC/Boc tert-Butoxycarbonyl;-   BSA Bovine Serum Albumin;-   CAN Ceric Ammonia Nitrate;-   CBz Carbobenzyloxy;-   CI Chemical Ionization;-   DBAD Di-tert-butyl azodicarboxylate;-   DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene;-   DCC 1,3 Dichlorohexylcarbodiimide;-   DCE 1,2-Dichloroethane;-   DCM Dichloromethane;-   DIEA N,N-Diisopropylethylamine;-   DMAP 4-Dimethylaminopyridine;-   DME 1,2-Dimethoxyethane;-   DMF N,N-Dimethylformamide;-   DMSO Methyl sulfoxide;-   DPPA Diphenylphosphoryl azide;-   DTT Dithiothreitol;-   EDC 1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide-hydrochloride;-   EDTA Ethylenediaminetetraacetic acid;-   ELSD Evaporative Light Scattering Detector;-   ES Electrospray;-   ESI Electrospray ionization;-   Et₂O Diethyl ether;-   Et₃N Triethylamine;-   EtOAc Ethyl acetate;-   EtOH Ethanol;-   FAB Fast atom bombardment;-   HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid;-   HMPA Hexamethylphosphoramide;-   HOAc Acetic acid;-   HOBT 1-Hydroxybenzotriazole hydrate;-   HOOBT 3-Hydroxy-1,2,2-benzotriazin-4(3H)-one;-   HPLC High-performance liquid chromatography;-   HRMS High Resolution Mass Spectroscopy;-   KOtBu Potassium tert-butoxide;-   LAH Lithium aluminum hydride;-   LCMS Liquid Chromatography Mass Spectroscopy;-   MCPBA m-Chloroperoxybenzoic acid;-   Me Methyl;-   MeOH Methanol;-   Ms Methanesulfonyl;-   MS Mass Spectroscopy;-   MsCl Methanesulfonyl chloride;-   n-Bu n-butyl;-   n-Bu₃P Tri-n-butylphosphine;-   NaHMDS Sodium bis(trimethylsilyl)amide;-   NBS N-Bromosuccinimide;-   NMM N-methylmorpholine;-   NMR Nuclear Magnetic Resonance;-   Pd(PPh₃)₄ Palladium tetrakis(triphenylphosphine);-   Pd₂(dba)₃ Tris(dibenzylideneacetone)dipalladium (0)-   Ph Phenyl;-   PMSF α-Toluenesulfonyl fluoride;-   PS-DCC Polystyrene dicyclohexylcarbodiimide;-   PS-DMAP Polystyrene dimethylaminopyridine;-   PS-NMM Polystyrene N-methylmorpholine;-   Py or pyr Pyridine;-   PYBOP Benzotriazol-1-yloxytripyrrolidinophosphonium-   (or PyBOP) hexafluorophosphate;-   RPLC Reverse Phase Liquid Chromatography;-   RT Room Temperature;-   SCX SPE Strong Cation Exchange Solid Phase Extraction;-   t-Bu tert-Butyl;-   TBAF Tetrabutylammonium fluoride;-   TBSCI tert-Butyldimethylsilyl chloride;-   TFA Trifluoroacetic acid;-   THF Tetrahydrofuran;-   TIPS Triisopropylsilyl;-   TMS Tetramethylsilane; and-   Tr Trityl.

The compounds of this invention may be prepared by employing reactionsas shown in the following schemes, in addition to other standardmanipulations that are known in the literature or exemplified in theexperimental procedures. These schemes, therefore, are not limited bythe compounds listed nor by any particular substituents employed forillustrative purposes. Substituent numbering as shown in the schemes donot necessarily correlate to that used in the claims.

Utility

The instant compounds are useful as pharmaceutical agents for mammals,especially for humans, in the treatment of tyrosine kinase dependentdiseases. Such diseases include the proliferation of tumor cells, thepathologic neovascularization (or angiogenesis) that supports solidtumor growth, ocular neovascularization (diabetic retinopathy,age-related macular degeneration, and the like) and inflammation(psoriasis, rheumatoid arthritis, and the like).

The compounds of the instant invention may be administered to patientsfor use in the treatment of cancer. The instant compounds inhibit tumorangiogenesis, thereby affecting the growth of tumors (J. Rak et al.Cancer Research, 55:4575-4580, 1995). The anti-angiogenesis propertiesof the instant compounds are also useful in the treatment of certainforms of blindness related to retinal vascularization.

The disclosed compounds are also useful in the treatment of certainbone-related pathologies, such as osteosarcoma, osteoarthritis, andrickets, also known as oncogenic osteomalacia. (Hasegawa et al.,Skeletal Radiol., 28, pp. 41-45, 1999; Gerber et al., Nature Medicine,Vol. 5, No. 6, pp. 623-628, June 1999). And since VEGF directly promotesosteoclastic bone resorption through KDR/Flk-1 expressed in matureosteoclasts (FEBS Let. 473:161-164 (2000); Endocrinology, 141:1667(2000)), the instant compounds are also useful to treat and preventconditions related to bone resorption, such as osteoporosis and Paget'sdisease.

The claimed compounds can also be used to reduce or prevent tissuedamage which occurs after cerebral ischemic events, such as stroke, byreducing cerebral edema, tissue damage, and reperfusion injury followingischemia. (Drug News Perspect 11:265-270 (1998); J. Clin. invest.104:1613-1620 (1999); Nature Med 7:222-227 (2001)).

The compounds of this invention may be administered to mammals,preferably humans, either alone or, preferably, in combination withpharmaceutically acceptable carriers or diluents, optionally with knownadjuvants, such as alum, in a pharmaceutical composition, according tostandard pharmaceutical practice. The compounds can be administeredorally or parenterally, including the intravenous, intramuscular,intraperitoneal, subcutaneous, rectal and topical routes ofadministration.

For oral use of a chemotherapeutic compound according to this invention,the selected compound may be administered, for example, in the form oftablets or capsules, or as an aqueous solution or suspension. In thecase of tablets for oral use, carriers which are commonly used includelactose and cornstarch, and lubricating agents, such as magnesiumstearate, are commonly added. For oral administration in capsule form,useful diluents include lactose and dried cornstarch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweeteningand/or flavoring agents may be added. For intramuscular,intraperitoneal, subcutaneous and intravenous use, sterile solutions ofthe active ingredient are usually prepared, and the pH of the solutionsshould be suitably adjusted and buffered. For intravenous use, the totalconcentration of solutes should be controlled in order to render thepreparation isotonic.

The instant compounds may also be co-administered with other well knowntherapeutic agents that are selected for their particular usefulnessagainst the condition that is being treated.

For example, the instant compounds are useful in combination with knownanti-cancer agents. Combinations of the presently disclosed compoundswith other anti-cancer or chemotherapeutic agents are within the scopeof the invention. Examples of such agents can be found in CancerPrinciples and Practice of Oncology by V. T. Devita and S. Hellman(editors), 6^(th) edition Feb. 15, 2001), Lippincott Williams & WilkinsPublishers. A person of ordinary skill in the art would be able todiscern which combinations of agents would be useful based on theparticular characteristics of the drugs and the cancer involved. Suchanti-cancer agents include the following: estrogen receptor modulators,androgen receptor modulators, retinoid receptor modulators,cytotoxic/cytostatic agents, antiproliferative agents, prenyl-proteintransferase inhibitors, HMG-CoA reductase inhibitors and otherangiogenesis inhibitors and agents that interfere with cell cyclecheckpoints. The instant compounds are particularly useful whenco-administered with radiation therapy. The synergistic effects ofinhibiting VEGF in combination with radiation therapy have beendescribed in the art. (See WO 00/61186).

“Antiproliferative agents” also includes monoclonal antibodies to growthfactors, other than those listed under “angiogenesis inhibitors”, suchas trastuzumab, and tumor suppressor genes, such as p53, which can bedelivered via recombinant virus-mediated gene transfer (see U.S. Pat.No. 6,069,134, for example).

“Estrogen receptor modulators” refers to compounds that interfere withor inhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,α-difluoromethylornithine, ILX23-7553, trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell deathor inhibit cell proliferation primarily by interfering directly with thecell's functioning or inhibit or interfere with cell myosis, includingalkylating agents, tumor necrosis factors, intercalators, hypoxiaactivatable compounds, microtubule inhibitors/microtubule-stabilizingagents, inhibitors of mitotic kinesins, anti-metabolites; biologicalresponse modifiers; hormonal/anti-hormonal therapeutic agents,haematopoietic growth factors, monoclonal antibody targeted therapeuticagents and topoisomerase inhibitors.

Examples of cytotoxic agents include, but are not limited to, sertenef,cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine,prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin,oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfantosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa,lobaplatin, satraplatin, profiromycin, cisplatin, irofulven,dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032).

An example of a hypoxia activatable compound is tirapazamine.

Examples of microtubule inhibitors/microtubule-stabilising agentsinclude paclitaxel, vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol, rhizoxin,dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237) and BMS188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′—O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine,(5a,5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, are described in PCT Publications WO 01/30768 andWO 01/98278, and pending U.S. Ser. Nos. 60/338,779 (filed Dec. 6, 2001),60/338,344 (filed Dec. 6, 2001), 60/338,383 (filed Dec. 6, 2001),60/338,380 (filed Dec. 6, 2001), 60/338,379 (filed Dec. 6, 2001) and60/344,453 (filed Nov. 7, 2001).

“Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-flurouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N-4-palmitoyl-1-B-D-arabino furanosyl cytosine,3-aminopyridine-2-carboxaldehyde thiosemicarbazone and trastuzumab.

Examples of monoclonal antibody targeted therapeutic agents includethose therapeutic agents which have cytotoxic agents or radioisotopesattached to a cancer cell specific or target cell specific monoclonalantibody. Examples include Bexxar.

“HWG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Compounds which haveinhibitory activity for HMG-CoA reductase can be readily identified byusing assays well-known in the art. For example, see the assaysdescribed or cited in U.S. Pat. No. 4,231,938 at col. 6, and WO 84/02131at pp. 30-33. The terms “HMG-CoA reductase inhibitor” and “inhibitor ofHMG-CoA reductase” have the same meaning when used herein.

Examples of HMG-CoA reductase inhibitors that may be used include butare not limited to lovastatin (MEVACOR®; see U.S. Pat. Nos.4,231,938,4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Pat.Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; seeU.S. Pat. Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and5,180,589), fluvastatin (LESCOL®; see U.S. Pat. Nos. 5,354,772,4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896),atorvastatin (LIPITOR®; see U.S. Pat. Nos. 5,273,995,4,681,893,5,489,691 and 5,342,952) and cerivastatin (also known as rivastatin andBAYCHOL®; see U.S. Pat. No. 5,177,080). The structural formulas of theseand additional HMG-CoA reductase inhibitors that may be used in theinstant methods are described at page 87 of M. Yalpani, “CholesterolLowering Drugs”, Chemistry & Industry, pp. 85-89 (5 Feb. 1996) and U.S.Pat. Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitoras used herein includes all pharmaceutically acceptable lactone andopen-acid forms (i.e., where the lactone ring is opened to form the freeacid) as well as salt and ester forms of compounds which have HMG-CoAreductase inhibitory activity, and therefor the use of such salts,esters, open-acid and lactone forms is included within the scope of thisinvention. An illustration of the lactone portion and its correspondingopen-acid form is shown below as structures I and II.

In HMG-CoA reductase inhibitors where an open-acid form can exist, saltand ester forms may preferably be formed from the open-acid, and allsuch forms are included within the meaning of the term “HMG-CoAreductase inhibitor” as used herein. Preferably, the HMG-CoA reductaseinhibitor is selected from lovastatin and simvastatin, and mostpreferably simvastatin. Herein, the term “pharmaceutically acceptablesalts” with respect to the HMG-CoA reductase inhibitor shall meannon-toxic salts of the compounds employed in this invention which aregenerally prepared by reacting the free acid with a suitable organic orinorganic base, particularly those formed from cations such as sodium,potassium, aluminum, calcium, lithium, magnesium, zinc andtetramethylammonium, as well as those salts formed from amines such asammonia, ethylenediamine, N-methylglucamine, lysine, arginine,ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine,diethanolamine, procaine, N-benzylphenethylamine,1-p-chlorobenzyl-2-pyrrolidine-1′-yl-methylbenz-imidazole, diethylamine,piperazine, and tris(hydroxymethyl) aminomethane. Further examples ofsalt forms of HMG-CoA reductase inhibitors may include, but are notlimited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,bitartrate, borate, bromide, calcium edetate, camsylate, carbonate,chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynapthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote,palmitate, panthothenate, phosphate/diphosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, and valerate.

Ester derivatives of the described HMG-CoA reductase inhibitor compoundsmay act as prodrugs which, when absorbed into the bloodstream of awarm-blooded animal, may cleave in such a manner as to release the drugform and permit the drug to afford improved therapeutic efficacy.

“Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase). Examples of prenyl-protein transferase inhibiting compoundsinclude(±)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,(−)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,(+)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,5(S)-n-butyl-1-(2,3-dimethylphenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,(S)-1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-5-[2-(ethanesulfonyl)methyl)-2-piperazinone,5(S)-n-Butyl-1-(2-methylphenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-2-methyl-5-imidazolylmethyl]-2-piperazinone,1-(2,2-diphenylethyl)-3-[N-(1-(4-cyanobenzyl)-1H-imidazol-5-ylethyl)carbamoyl]piperidine,4-{5-[4-hydroxymethyl-4-(4-chloropyridin-2-ylmethyl)-piperidine-1-ylmethyl]-2-methylimidazol-1-ylmethyl}benzonitrile,4-{5-[4-hydroxymethyl-4-(3-chlorobenzyl)-piperidine-1-ylmethyl]-2-methylimidazol-1-ylmethyl}benzonitrile,4-{3-[4-(2-oxo-2H-pyridin-1-yl)benzyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-{3-[4-(5-chloro-2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-{3-[4-(2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-[3-(2-oxo-1-phenyl-1,2-dihydropyridin-4-ylmethyl)-3H-imidazol-4-ylmethyl}benzonitrile,18,19-dihydro-19-oxo-5H,17H-6,10:12,16-dimetheno-1H-imidazo[4,3-c][1,11,4]dioxaazacyclo-nonadecine-9-carbonitrile,(±)-19,20-dihydro-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriaza-cyclooctadecine-9-carbonitrile,19,20-dihydro-19-oxo-5H,17H-18,21-ethano-6,10:12,16-dimetheno-22H-imidazo[3,4-h][1,8,11,14]oxatriazacycloeicosine-9-carbonitrile,and(+)-19,20-dihydro-3-methyl-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxa-triazacyclooctadecine-9-carbonitrile.

Other examples of prenyl-protein transferase inhibitors can be found inthe following publications and patents: WO 96/30343, WO 97/18813, WO97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO95/32987, U.S. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430, U.S. Pat.No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No. 5,589,485, U.S.Pat. No. 5,602,098, European Patent Publ. 0 618 221, European PatentPubl. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ.0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of aprenyl-protein transferase inhibitor on angiogenesis see European J. ofCancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

“Angiogenesis inhibitors” refers to compounds that inhibit the formationof new blood vessels, regardless of mechanism. Examples of angiogenesisinhibitors include, but are not limited to, tyrosine kinase inhibitors,such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) andFlk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived,or platelet derived growth factors, MMP (matrix metalloprotease)inhibitors, integrin blockers, interferon-α, interleukin-12, pentosanpolysulfate, cyclooxygenase inhibitors, including nonsteroidalanti-inflammatories (NSAIDs) like aspirin and ibuprofen as well asselective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib(PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch.Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68 (1994);FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76(1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol.,Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol.93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol.Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such ascorticosteroids, mineralocorticoids, dexamethasone, prednisone,prednisolone, methylpred, betamethasone), carboxyamidotriazole,combretastatin A4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, troponin-1, angiotensin II antagonists (seeFernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodiesto VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968 (October 1999);Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be used in combination with the compounds of the instant inventioninclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354(2001)). TAFIa inhibitors have been described in U.S. Ser. Nos.60/310,927 (filed Aug. 8, 2001) and 60/349,925 (filed Jan. 18, 2002).

“Agents that interfere with cell cycle checkpoints” refer to compoundsthat inhibit protein kinases that transduce cell cycle checkpointsignals, thereby sensitizing the cancer cell to DNA damaging agents.Such agents include inhibitors of ATR, ATM, the Chk1 and Chk2 kinasesand cdk and cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

As described above, the combinations with NSAID's are directed to theuse of NSAID's which are potent COX-2 inhibiting agents. For purposes ofthis specification an NSAID is potent if it possess an IC₅₀ for theinhibition of COX-2 of 1 μM or less as measured by cell or microsomalassays.

Additionally, in the case of bone-related disorders, combinations thatwould be useful include those with antiresorptive bisphosphonates, suchas alendronate and risedronate; integrin blockers (defined furtherbelow), such as α_(v)β₃ antagonists; conjugated estrogens used inhormone replacement therapy, such as PREMPRO®, PREMARIN® andENDOMTRION®; selective estrogen receptor modulators (SERMs), such asraloxifene, droloxifene, CP-336,156 (Pfizer) and lasofoxifene; cathespinK inhibitors; and ATP proton pump inhibitors.

The invention also encompasses combinations with NSAID's which areselective COX-2 inhibitors. For purposes of this specification NSAID'swhich are selective inhibitors of COX-2 are defined as those whichpossess a specificity for inhibiting COX-2 over COX-1 of at least 100fold as measured by the ratio of IC₅₀ for COX-2 over IC₅₀ for COX-1evaluated by cell or microsomal assays. Such compounds include, but arenot limited to those disclosed in U.S. Pat. No. 5,474,995, issued Dec.12, 1995, U.S. Pat. No. 5,861,419, issued Jan. 19, 1999, U.S. Pat. No.6,001,843, issued Dec. 14, 1999, U.S. Pat. No. 6,020,343, issued Feb. 1,2000, U.S. Pat. No. 5,409,944, issued Apr. 25, 1995, U.S. Pat. No.5,436,265, issued Jul. 25, 1995, U.S. Pat. No. 5,536,752, issued Jul.16, 1996, U.S. Pat. No. 5,550,142, issued Aug. 27, 1996, U.S. Pat. No.5,604,260, issued Feb. 18, 1997, U.S. Pat. No. 5,698,584, issued Dec.16, 1997, U.S. Pat. No. 5,710,140, issued Jan. 20, 1998, WO 94/15932,published Jul. 21, 1994, U.S. Pat. No. 5,344,991, issued Jun. 6, 1994,U.S. Pat. No. 5,134,142, issued Jul. 28, 1992, U.S. Pat. No. 5,380,738,issued Jan. 10, 1995, U.S. Pat. No. 5,393,790, issued Feb. 20, 1995,U.S. Pat. No. 5,466,823, issued Nov. 14, 1995, U.S. Pat. No. 5,633,272,issued May 27, 1997, and U.S. Pat. No. 5,932,598, issued Aug. 3, 1999,all of which are hereby incorporated by reference.

Inhibitors of COX-2 that are particularly useful in the instant methodof treatment are:

-   3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and

-   5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine;

or a pharmaceutically acceptable salt thereof.

General and specific synthetic procedures for the preparation of theCOX-2 inhibitor compounds described above are found in U.S. Pat. No.5,474,995, issued Dec. 12, 1995, U.S. Pat. No. 5,861,419, issued Jan.19, 1999, and U.S. Pat. No. 6,001,843, issued Dec. 14, 1999, all ofwhich are herein incorporated by reference.

Compounds that have been described as specific inhibitors of COX-2 andare therefore useful in the present invention include, but are notlimited to, the following:

or a pharmaceutically acceptable salt thereof.

Compounds which are described as specific inhibitors of COX-2 and aretherefore useful in the present invention, and methods of synthesisthereof, can be found in the following patents, pending applications andpublications, which are herein incorporated by reference: WO 94/15932,published Jul. 21, 1994, U.S. Pat. No. 5,344,991, issued Jun. 6, 1994,U.S. Pat. No. 5,134,142, issued Jul. 28, 1992, U.S. Pat. No. 5,380,738,issued Jan. 10, 1995, U.S. Pat. No. 5,393,790, issued Feb. 20, 1995,U.S. Pat. No. 5,466,823, issued Nov. 14, 1995, U.S. Pat. No. 5,633,272,issued May 27, 1997, and U.S. Pat. No. 5,932,598, issued Aug. 3, 1999.

Compounds which are specific inhibitors of COX-2 and are thereforeuseful in the present invention, and methods of synthesis thereof, canbe found in the following patents, pending applications andpublications, which are herein incorporated by reference: U.S. Pat. No.5,474,995, issued Dec. 12, 1995, U.S. Pat. No. 5,861,419, issued Jan.19, 1999, U.S. Pat. No. 6,001,843, issued Dec. 14, 1999, U.S. Pat. No.6,020,343, issued Feb. 1, 2000, U.S. Pat. No. 5,409,944, issued Apr. 25,1995, U.S. Pat. No. 5,436,265, issued Jul. 25, 1995, U.S. Pat. No.5,536,752, issued Jul. 16, 1996, U.S. Pat. No. 5,550,142, issued Aug.27, 1996, U.S. Pat. No. 5,604,260, issued Feb. 18, 1997, U.S. Pat. No.5,698,584, issued Dec. 16, 1997, and U.S. Pat. No. 5,710,140, issuedJan. 20, 1998.

Other examples of angiogenesis inhibitors include, but are not limitedto, endostatin, ukrain, ranpimase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101,squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaosephosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

As used above, “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe α_(v)β₃ integrin, to compounds which selectively antagonize, inhibitor counteract binding of a physiological ligand to the αvβ5 integrin, tocompounds which antagonize, inhibit or counteract binding of aphysiological ligand to both the α_(v)β₃ integrin and the α_(v)β₅integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of the α_(v)β₆,α_(v)β₈, α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins. The term also refersto antagonists of any combination of α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈,α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins.

Some specific examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH268, genistein, STI571, CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant methods. For example, combinations of theinstantly claimed compounds with PPAR-γ (i.e., PPAR-gamma) agonists andPPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment ofcertain malingnancies. PPAR-γ and PPAR-δ are the nuclear peroxisomeproliferator-activated receptors γ and δ. The expression of PPAR-γ onendothelial cells and its involvement in angiogenesis has been reportedin the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J.Biol. Chem. 1999;274:9116-9121; Invest. Ophthalmol Vis. Sci. 2000;41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibitthe angiogenic response to VEGF in vitro; both troglitazone androsiglitazone maleate inhibit the development of retinalneovascularization in mice. (Arch. Ophthanwl. 2001; 119:709-717).Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are notlimited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone,rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate,GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544,NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid (disclosed in U.S.Ser. Nos. 60/235,708 and 60/244,697).

Another embodiment of the instant invention is the use of the presentlydisclosed compounds in combination with gene therapy for the treatmentof cancer. For an overview of genetic strategies to treating cancer seeHall et al (Am J Hum Genet 61:785-789, 1997) and Kufe et al (CancerMedicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapycan be used to deliver any tumor suppressing gene. Examples of suchgenes include, but are not limited to, p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of auPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth andDissemination in Mice,” Gene Therapy, August 1998;5(8):1105-13), andinterferon gamma (J Immunol 2000;164:217-222).

The compounds of the instant invention may also be administered incombination with an inhibitor of inherent multidrug resistance (MDR), inparticular MDR associated with high levels of expression of transporterproteins. Such MDR inhibitors include inhibitors of p-glycoprotein(P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833(valspodar).

A compound of the present invention may be employed in conjunction withanti-emetic agents to treat nausea or emesis, including acute, delayed,late-phase, and anticipatory emesis, which may result from the use of acompound of the present invention, alone or with radiation therapy. Forthe prevention or treatment of emesis, a compound of the presentinvention may be used in conjunction with other anti-emetic agents,especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists,such as ondansetron, granisetron, tropisetron, and zatisetron, GABABreceptor agonists, such as baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten orothers such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401,3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, anantidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol. For the treatment or prevention of emesisthat may result upon administration of the instant compounds,conjunctive therapy with an anti-emesis agent selected from aneurokinin-1 receptor antagonist, a 5HT3 receptor antagonist and acorticosteroid is preferred.

Neurokinin-1 receptor antagonists of use in conjunction with thecompounds of the present invention are fully described, for example, inU.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595,5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European PatentPublication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0545 478, 0 558 156, 0 577 394, 0 585 913, 0 590 152, 0 599 538, 0 610793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733632 and 0 776 893; PCT International Patent Publication Nos. WO90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151,92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330,93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116,93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181,93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429,94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165,94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767,94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309,95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549,95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129,95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418,95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094,96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304,96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553,97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084,97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529,2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293169, and 2 302 689. The preparation of such compounds is fully describedin the aforementioned patents and publications, which are incorporatedherein by reference.

A particularly preferred neurokinin-1 receptor antagonist for use inconjunction with the compounds of the present invention is2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine,or a pharmaceutically acceptable salt thereof, which is described inU.S. Pat. No. 5,719,147.

A compound of the instant invention may also be administered with anagent useful in the treatment of anemia. Such an anemia treatment agentis, for example, a continuous eythropoiesis receptor activator (such asepoetin alfa).

A compound of the instant invention may also be administered with anagent useful in the treatment of neutropenia. Such a neutropeniatreatment agent is, for example, a hematopoietic growth factor whichregulates the production and function of neutrophils such as a humangranulocyte colony stimulating factor, (G-CSF). Examples of a G-CSFinclude filgrastim.

A compound of the instant invention may also be administered with animmunologic-enhancing drug, such as levamisole, isoprinosine andZadaxin.

Thus, the scope of the instant invention encompasses the use of theinstantly claimed compounds in combination with a second compoundselected from:

1) an estrogen receptor modulator,

2) an androgen receptor modulator,

3) retinoid receptor modulator,

4) a cytotoxic agent,

5) an antiproliferative agent,

6) a prenyl-protein transferase inhibitor,

7) an HMG-CoA reductase inhibitor,

8) an HIV protease inhibitor,

9) a reverse transcriptase inhibitor,

10) an angiogenesis inhibitor,

11) PPAR-γ agonists,

12) PPAR-δ agonists,

13) an inhibitor of inherent multidrug resistance,

14) an antiemetic agent,

15) an agent useful in the treatment of anemia,

16) agent useful in the treatment of neutropenia, and

17) an immunologic-enhancing drug.

Preferred angiogenesis inhibitors to be used as the second compound area tyrosine kinase inhibitor, an inhibitor of epidermal-derived growthfactor, an inhibitor of fibroblast-derived growth factor, an inhibitorof platelet derived growth factor, an MMP (matrix metalloprotease)inhibitor, an integrin blocker, interferon-α, interleukin-12, pentosanpolysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, troponin-1, or an antibody to VEGF. Preferredestrogen receptor modulators are tamoxifen and raloxifene.

Also included in the scope of the claims is a method of treating cancerthat comprises administering a therapeutically effective amount of acompound of Formula I in combination with radiation therapy and/or incombination with a compound selected from:

-   -   1) an estrogen receptor modulator,    -   2) an androgen receptor modulator,    -   3) retinoid receptor modulator,    -   4) a cytotoxic agent,    -   5) an antiproliferative agent,    -   6) a prenyl-protein transferase inhibitor,    -   7) an HMG-CoA reductase inhibitor,    -   8) an HIV protease inhibitor,    -   9) a reverse transcriptase inhibitor,    -   10) an angiogenesis inhibitor,    -   11) PPAR-γ agonists,    -   12) PPAR-δ agonists,    -   13) an inhibitor of inherent multidrug resistance,    -   14) an anti-emetic agent,    -   15) an agent useful in the treatment of anemia,    -   16) agent useful in the treatment of neutropenia, and    -   17) an immunologic-enhancing drug.

And yet another embodiment of the invention is a method of treatingcancer that comprises administering a therapeutically effective amountof a compound of Formula I in combination with paclitaxel ortrastuzumab.

The invention further encompasses a method of treating or preventingcancer that comprises administering a therapeutically effective amountof a compound of Formula I in combination with a COX-2 inhibitor.

The instant invention also includes a pharmaceutical composition usefulfor treating or preventing cancer that comprises a therapeuticallyeffective amount of a compound of Formula I and a compound selectedfrom:

-   -   1) an estrogen receptor modulator,    -   2) an androgen receptor modulator,    -   3) retinoid receptor modulator,    -   4) a cytotoxic agent,    -   5) an antiproliferative agent,    -   6) a prenyl-protein transferase inhibitor,    -   7) an HMG-CoA reductase inhibitor,    -   8) an HIV protease inhibitor,    -   9) a reverse transcriptase inhibitor,    -   10) an angiogenesis inhibitor, and    -   11) a PPAR-γ agonist, and    -   12) PPAR-δ agonists.

Examples of HIV protease inhibitors include amprenavir, abacavir,CGP-73547, CGP-61755, DMPA-450, indinavir, nelfinavir, tipranavir,ritonavir, saquinavir, ABT-378, AG 1776, and BMS-232,632. Examples ofreverse transcriptase inhibitors include delaviridine, efavirenz,GS-840, HB Y097, lamivudine, nevirapine, AZT, 3TC, ddC, and ddI.

The instant compounds are also useful, alone or in combination withplatelet fibrinogen receptor (GP IIb/IIIa) antagonists, such astirofiban, to inhibit metastasis of cancerous cells. Tumor cells canactivate platelets largely via thrombin generation. This activation isassociated with the release of VEGF. The release of VEGF enhancesmetastasis by increasing extravasation at points of adhesion to vascularendothelium (Amirkhosravi, Platelets 10, 285-292, 1999). Therefore, thepresent compounds can serve to inhibit metastasis, alone or incombination with GP IIb/IIa) antagonists. Examples of other fibrinogenreceptor antagonists include abciximab, eptifibatide, sibrafiban,lamifiban, lotrafiban, cromofiban, and CT50352.

If formulated as a fixed dose, such combination products employ thecompounds of this invention within the dosage range described below andthe other pharmaceutically active agent(s) within its approved dosagerange. Compounds of the instant invention may alternatively be usedsequentially with known pharmaceutically acceptable agent(s) when acombination formulation is inappropriate.

The term “administration” and variants thereof (e.g., “administering” acompound) in reference to a compound of the invention means introducingthe compound or a prodrug of the compound into the system of the animalin need of treatment. When a compound of the invention or prodrugthereof is provided in combination with one or more other active agents(e.g., a cytotoxic agent, etc.), “administration” and its variants areeach understood to include concurrent and sequential introduction of thecompound or prodrug thereof and other agents.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician.

The term “treating cancer” or “treatment of cancer” refers toadministration to a mammal afflicted with a cancerous condition andrefers to an effect that alleviates the cancerous condition by killingthe cancerous cells, but also to an effect that results in theinhibition of growth and/or metastasis of the cancer.

The present invention also encompasses a pharmaceutical compositionuseful in the treatment of cancer, comprising the administration of atherapeutically effective amount of the compounds of this invention,with or without pharmaceutically acceptable carriers or diluents.Suitable compositions of this invention include aqueous solutionscomprising compounds of this invention and pharmacologically acceptablecarriers, e.g., saline, at a pH level, e.g., 7.4. The solutions may beintroduced into a patient's bloodstream by local bolus injection.

When a compound according to this invention is administered into a humansubject, the daily dosage will normally be determined by the prescribingphysician with the dosage generally varying according to the age,weight, and response of the individual patient, as well as the severityof the patient's symptoms.

In one exemplary application, a suitable amount of compound isadministered to a mammal undergoing treatment for cancer. Administrationoccurs in an amount between about 0.1 mg/kg of body weight to about 60mg/kg of body weight per day, preferably of between 0.5 mg/kg of bodyweight to about 40 mg/kg of body weight per day.

Assays

The compounds of the instant invention described in the Examples weretested by the assays described below and were found to have kinaseinhibitory activity. Other assays are known in the literature and couldbe readily performed by those of skill in the art. (see, for example,Dhanabal et al., Cancer Res. 59:189-197; Xin et al., J. Biol. Chem.274:9116-9121; Sheu et al., Anticancer Res. 18:4435-4441; Ausprunk etal., Dev. Biol. 38:237-248; Gimbrone et al., J. Natl. Cancer Inst.52:413-427; Nicosia et al., In Vitro 18:538-549.)

I. VEGF Receptor Kinase Assay

VEGF receptor kinase activity is measured by incorporation ofradio-labeled phosphate into polyglutamic acid, tyrosine, 4:1 (pEY)substrate. The phosphorylated pEY product is trapped onto a filtermembrane and the incorporation of radio-labeled phosphate quantified byscintillation counting.

Materials

VEGF Receptor Kinase

The intracellular tyrosine kinase domains of human KDR (Terman, B. I. etal. Oncogene (1991) vol. 6, pp. 1677-1683.) and Flt-1 (Shibuya, M. etal. Oncogene (1990) vol. 5, pp. 519-524) were cloned as glutathioneS-transferase (GST) gene fusion proteins. This was accomplished bycloning the cytoplasmic domain of the KDR kinase as an in frame fusionat the carboxy terminus of the GST gene. Soluble recombinant GST-kinasedomain fusion proteins were expressed in Spodoptera frugiperda (Sf21)insect cells (Invitrogen) using a baculovirus expression vector (pAcG2T,Pharmingen).

The other materials used and their compositions were as follows:

-   Lysis buffer: 50 mM Tris pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mM EDTA,    0.5% triton X-100, 10% glycerol, 10 mg/mL of each leupeptin,    pepstatin and aprotinin and 1 mM phenylmethylsulfonyl fluoride (all    Sigma).-   Wash buffer: 50 mM Tris pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mM EDTA,    0.05% triton X-100, 10% glycerol, 10 mg/mL of each leupeptin,    pepstatin and aprotinin and 1 mM phenylmethylsulfonyl fluoride.-   Dialysis buffer: 50 mM Tris pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mM EDTA,    0.05% triton X-100, 50% glycerol, 10 mg/mL of each leupeptin,    pepstatin and aprotinin and 1 mM phenylmethylsuflonyl fluoride.-   10× reaction buffer: 200 mM Tris, pH 7.4, 1.0 M NaCl, 50 mM MnCl₂,    10 mM DTT and 5 mg/mL bovine serum albumin (Sigma).-   Enzyme dilution buffer: 50 mM Tris, pH 7.4, 0.1 M NaCl, 1 mM DTT,    10% glycerol, 100 mg/mL BSA.-   10× Substrate: 750 μg/mL poly (glutamic acid, tyrosine; 4:1)    (Sigma).-   Stop solution: 30% trichloroacetic acid, 0.2 M sodium pyrophosphate    (both Fisher).-   Wash solution: 15% trichloroacetic acid, 0.2 M sodium pyrophosphate.-   Filter plates: Millipore #MAFC NOB, GF/C glass fiber 96 well plate.

Method

A. Protein purification

1. Sf21 cells were infected with recombinant virus at a multiplicity ofinfection of 5 virus particles/cell and grown at 27° C. for 48 hours.

2. All steps were performed at 4° C. Infected cells were harvested bycentrifugation at 1000×g and lysed at 4° C. for 30 minutes with 1/10volume of lysis buffer followed by centrifugation at 100,000×g for 1hour. The supernatant was then passed over a glutathione Sepharosecolumn (Pharmacia) equilibrated in lysis buffer and washed with 5volumes of the same buffer followed by 5 volumes of wash buffer.Recombinant GST-KDR protein was eluted with wash buffer/10 mM reducedglutathione (Sigma) and dialyzed against dialysis buffer.

B. VEGF Receptor Kinase Assay

1. Add 5 μl, of inhibitor or control to the assay in 50% DMSO.

2. Add 35 μl of reaction mix containing 5 μl of 10× reaction buffer, 5μl 25 mM ATP/10 μCi [³³P]ATP (Amersham), and 5 μl 10× substrate.

3. Start the reaction by the addition of 10 μl of KDR (25 nM) in enzymedilution buffer.

4. Mix and incubate at room temperature for 15 minutes.

5. Stop by the addition of 50 μl stop solution.

6. Incubate for 15 minutes at 4° C.

7. Transfer a 90 μl, aliquot to filter plate.

8. Aspirate and wash 3 times with wash solution.

9. Add 30 μl of scintillation cocktail, seal plate and count in a WallacMicrobeta scintillation counter.

II. Human Umbilical Vein Endothelial Cell Mitogenesis Assay

Human umbilical vein endothelial cells (HUVECs) in culture proliferatein response to VEGF treatment and can be used as an assay system toquantify the effects of KDR kinase inhibitors on VEGF stimulation. Inthe assay described, quiescent HUVEC monolayers are treated with vehicleor test compound 2 hours prior to addition of VEGF or basic fibroblastgrowth factor (bFGF). The mitogenic response to VEGF or bFGF isdetermined by measuring the incorporation of [³H]thymidine into cellularDNA.

Materials

-   HUVECs: HUVECs frozen as primary culture isolates are obtained from    Clonetics Corp. Cells are maintained in Endothelial Growth Medium    (EGM; Clonetics) and are used for mitogenic assays described in    passages 3-7 below.-   Culture Plates: NUNCLON 96-well polystyrene tissue culture plates    (NUNC #167008).-   Assay Medium: Dulbecco's modification of Eagle's medium containing 1    g/mL glucose (low-glucose DMEM; Mediatech) plus 10% (v/v) fetal    bovine serum (Clonetics).-   Test Compounds: Working stocks of test compounds are diluted    serially in 100% dimethylsulfoxide (DMSO) to 400-fold greater than    their desired final concentrations. Final dilutions to 1×    concentration are made directly into Assay Medium immediately prior    to addition to cells.-   10× Growth Factors: Solutions of human VEGF₁₆₅ (500 ng/mL; R&D    Systems) and bFGP (10 ng/mL; R&D Systems) are prepared in Assay    Medium.-   10×[³H]Thymidine: [Methyl-³H]thymidine (20 Ci/mmol; Dupont-NEN) is    diluted to 80 μCi/mL in low-glucose DMEM.-   Cell Wash Medium: Hank's balanced salt solution (Mediatech)    containing 1 mg/mL bovine serum albumin (Boehringer-Mannheim).-   Cell Lysis Solution: 1 N NaOH, 2% (w/v) Na₂CO₃.

Method

1. HUVEC monolayers maintained in EGM are harvested by trypsinizationand plated at a density of 4000 cells per 100 μL Assay Medium per wellin 96-well plates. Cells are growth-arrested for 24 hours at 37° C. in ahumidified atmosphere containing 5% CO₂.

2. Growth-arrest medium is replaced by 100 μL Assay Medium containingeither vehicle (0.25% [v/v] DMSO) or the desired final concentration oftest compound. All determinations are performed in triplicate. Cells arethen incubated at 37° C. with 5% CO₂ for 2 hours to allow test compoundsto enter cells.

3. After the 2-hour pretreatment period, cells are stimulated byaddition of 10 μL/well of either Assay Medium, 10×VEGF solution or10×bFGF solution. Cells are then incubated at 37° C. and 5% CO₂.

4. After 24 hours in the presence of growth factors, 10×[³H] thymidine(10 μL/well) is added.

5. Three days after addition of [³H]thymidine, medium is removed byaspiration, and cells are washed twice with Cell Wash Medium (400μL/well followed by 200 μL/well). The washed, adherent cells are thensolubilized by addition of Cell Lysis Solution (100 μL/well) and warmingto 37° C. for 30 minutes. Cell lysates are transferred to 7-mL glassscintillation vials containing 150 μL of water. Scintillation cocktail(5 mL/vial) is added, and cell-associated radioactivity is determined byliquid scintillation spectroscopy.

Based upon the foregoing assays the compounds of Formula I areinhibitors of VEGF and thus are useful for the inhibition ofangiogenesis, such as in the treatment of ocular disease, e.g., diabeticretinopathy and in the treatment of cancers, e.g., solid tumors. Theinstant compounds inhibit VEGF-stimulated mitogenesis of human vascularendothelial cells in culture with IC₅₀ values between 0.001-5.0 μM.These compounds also show selectivity over related tyrosine kinases(e.g., FGFR1 and the Src family; for relationship between Src kinasesand VEGFR kinases, see Eliceiri et al., Molecular Cell, Vol. 4, pp.915-924, December 1999).

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be illustrative of the invention and not limiting of thereasonable scope thereof. The compounds of this invention may beprepared by employing reactions as shown in the following schemes, inaddition to other standard manipulations that are known in theliterature or exemplified in the experimental procedures. These schemes,therefore, are not limited by the compounds listed or by any particularsubstituents employed for illustrative purposes.

6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid ethylester (1-1)

A solution of 3-amino-4-carboxylic ethyl ester-pyrazole (500 mg, 3.2mmol, 1 equiv) and methoxyphenyl dialdehyde (690 mg, 3.9 mmol, 1.2equiv) in EtOH (20 mL) was heated at reflux for 2 hours. The precipitatewas filtered and air dried to afford6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid ethylester (1-1) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 9.05 (m,1H), 8.84 (m, 1H), 8.59 (s, 1H), 7.53 (d, 2H, J=8 Hz), 7.07 (d, 2H, J=8Hz), 4.45 (q, 2H, J=6.0 Hz), 3.89 (s, 1H), 1.44 (t, 3H, J=6 Hz).

6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (1-2)

A solution of6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid ethylester (1-1, 660 mg, 2.2 mmol, 1 equiv) and aquoeus sodium hydroxidesolution (1N, 11.1 mL, 11.1 mmol, 5.00 equiv) in-BuOH (45 mL) wasstirred for 24 hours at 100° C. The yellow solution was poured directlyinto aqueous 1N HCl solution (500 mL), and the resulting aqueous mixturewas extracted with chloroform (3×300 mL). The combined organic layerswere dried over sodium sulfate and concentrated to afford6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (1-2)as a white solid. ¹H NMR (400 MHz, DMSO) δ 12.38 (br s, 1H), 9.54 (m,1H), 9.16 (m, 1H), 8.60 (s, 1H), 7.84 (d, 2H, J=8 Hz), 7.12 (d, 2H, J=8Hz), 3.85 (s, 3H).

6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid amide(1-3)

To a solution of6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid(1-2,610 mg, 2.27 mmol, 1 equiv) in CH₂Cl₂ (35 mL) was added oxalylchloride (988 TL, 11.3 mmol, 5.00 equiv), and the resulting whitesuspension was stirred for 35 minutes, by which time, the reactionmixture had become a yellow solution. The solvent and the excess oxalylchloride was removed in vacuo. The white residue was suspended inchloroform (100 mL) and treated with NH₃ gas until a precipitate formed.The precipitate was filtered, washed with water, and air dried to afford6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid amide(1-3).

-   ¹H NMR (400 MHz, DMSO-d₆) δ 9.55 (m, 1H), 9.16 (m, 1H), 8.57 (s,    1H), 7.84 (d, 2H, J=8.8 Hz), 7.49 (br d, 2H), 7.11 (d, 2H, J=8.8    Hz), 3.84 (s, 3H).

6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carbonitrile (1-4)

A solution of6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid amide(1-3, 150 mg, 0.56 mmol, 1 equiv) in dioxane (50 mL) was heated toreflux. After the solid was solvated, Burgess reagent (134 mg, 0.56mmol, 1.0 equiv) was added. More Burgess reagent (4 equivalents) wasadded in three equal portions over 3 hours after which TLC indicated thestarting amide had been consumed. The solvent was removed in vacuo andthe yellow residue was partitioned between EtOAc (2×200 mL) and water(330 mL). The combined organic layers were dried over sodium sulfate andconcentrated. The yellow residue was then suspended in ether andfiltered to afford6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carbonitrile (1-4).

-   ¹H NMR (300 MHz, CDCl₃) δ 8.97 (d, 1H, J=2.4 Hz), 8.85 (d, 1H, J=2.4    Hz), 8.41 (s, 1H), 7.53 (d, 2H, J=8.7 Hz), 7.09 (d, 2H, J=8.7 Hz),    3.89 (s, 3H).

3-iodo-6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine (2-1)

A solution of 4-iodo-1H-pyrazol-5-amine (640 mg, 3.9 mmol, 1 equiv) and2-(4-methoxyphenyl)malonaldehyde (700 mg, 3.9 mmol, 1.0 equiv) inethanol (20 mL) was heated at 75° C. for 4 hours. The reaction mixturewas allowed to cool to 23° C., and the precipitate was filtered, washedwith cold methanol, and air dried to afford3-iodo-6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine (2-1) as a whitesolid.

ethyl 5-[6-(4-methoxyphenyl)pyrazolor[1,5-a]pyrimidin-3-yl]nicotinate(2-2)

A mixture of ethyl 5-bromonicotinate (500 mg, 2.17 mmol, 1 equiv),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi-1,3,2-dioxaborolane (662 mg, 2.61mmol, 1.20 equiv), potassium acetate (639 g, 6.51 mmol, 3.00 equiv), and1,1′-bis (diphenylphophino)-ferrocene)dichloropalladium (89 mg, 0.11mmol, 0.050 equiv) in DMF (5 mL) was heated at 80° C. for 16 hours. Themixture was partitioned between water (100 mL) and ethyl acetate (2×50mL). The organic layer was dried over sodium sulfate and concentrated. Asolution of the residue,3-iodo-6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine (2-1, 254 mg, 0.723mmol, 0.333 equiv), tetrakis(triphenylphosphine)palladium (50 mg, 0.043mmol, 0.020 equiv) in a mixture of aqueous 2M sodium carbonate solution(1 mL) and dioxane (10 mL) was heated at reflux for 16 hours. Thereaction was then partitioned between water (50 mL) and ethyl acetate(2×50 mL). The organic layer was dried over sodium sulfate andconcentrated. The residue was suspended in methanol and filtered to giveethyl 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]nicotinate(2-2) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 9.50 (d, 1H, J=2.2Hz), 9.12 (d, 1H, J=2.2 Hz), 8.96 (t, 1H, J=2.2 Hz), 8.88 (d, 1H, J=2.2Hz), 8.8.3 (d, 1H, J=2.4 Hz), 8.54 (s, 1H), 7.56 (d, 2H, J=8.8 Hz), 7.08(d, 2H, J=8.8 Hz), 4.48 (q, 2H, J=7.3 Hz), 3.90 (s, 3H), 1.46 (t, 3H,J=7.1 Hz).

5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-N-methylnicotinamide(2-3)

A mixture of ethyl5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]nicotinate (2-2, 150mg, 0.40 mmol, 1 equiv) and aqueous sodium hydroxide solution (1N, 1.0mL), 1.0 mmol, 2.5 equiv) in a mixture of t-BuOH (2 mL) and DME (2 mL)was heated at reflux for 1 hour. The reaction mixture was allowed tocool to 23° C., and then it was acidified with aqueous 1 N hydrogenchloride solution. The precipitate was filtered, washed with water, andair dried. Oxalyl chloride (150 TL, 1.7 mmol, 4.3 equiv) followed by acatalytic amount of DMF (4 TL) was then added to a suspension of thefiltered solid in dichloromethane (10 mL), and the resulting mixture wasstirred for 30 minutes, then concentrated. A suspension of one-tenth ofthis residue (approximately 15 mg, 0.040 mmol), methylaminehydrochloride (27 mg, 0.40 mmol, 10 equiv) and N,N-diisopropylethylamine(200 TL, 1.1 mmol, 30 equiv) in dichloromethane (3 mL) was stirred at23° C. for 30 minutes. The reaction mixture was partitioned betweenhalf-saturated aqueous sodium bicarbonate solution. The organic layerwas dried over sodium sulfate and concentrated. The residue wassuspended in methanol (3 mL) and filtered to give5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-N-methylnicotinamide(2-3) as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.49 (d, 1H, J=2.2Hz), 9.45 (d, 1H, J=2.2 Hz), 9.12 (d, 1H, J=2.2 Hz), 8.91 (s, 1H), 8.88(t, 1H, J=2.0 Hz), 8.83 (d, 1H, J=2.0 Hz), 8.66 (br m, 1H), 7.84 (d, 2H,J=8.8 Hz), 7.10 (d, 2H, J=8.8 Hz), 3.82 (s, 3H), 2.84 (d, 3H, J=4.4 Hz).

The following compounds were prepared by simple modifications of theabove procedure.

Com- pound Name Structure 2-4 N-ethyl-5-[6-(4-meth-oxyphenyl)pyrazolo[1,5-a]py- rimidin-3-yl]nicotinamide

2-5 N-cyclopropyl-5-[6-(4-methoxyphenyl)py-razolo[1,5-a]pyrimidin-3-yl]nico- tinamide

2-6 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]py-rimidin-3-yl]-N-propylnicotinamide

2-7 5-[6-(3-methoxyphenyl)pyrazolo[1,5-a]py-rimidin-3-yl]-N-methylnicotinamide

2-8 N-ethyl-5-[6-(3-meth- oxyphenyl)pyrazolo[1,5-a]py-rimidin-3-yl]nicotinamide

2-9 5-[6-(3-methoxyphenyl)pyrazolo[1,5-a]py-rimidin-3-yl]-N-propylnicotinamide

2-10 N-cyclopropyl-5-(6-py- ridin-4-ylpyrazolo[1,5-a]py-rimidin-3-yl)nicotinamide

2-11 N-propyl-5-(6-py- ridin-4-ylpyrazolo[1,5-a]py-rimidin-3-yl)nicotinamide

1. A compound of formula I

wherein m is independently 0,1 or 2; R¹ is: 1) C₂-C₆ alkenyl, 2) C₂-C₆alkynyl, 3) C₂-C₈ alkyl, 4) (C═O)NR^(a)R^(b), 5) (C═O)R^(c) 6)(C═O)OR^(c), or 7) heterocyclyl, said heterocyclyl is substituted withat least one substituent selected from: a) C₀-C₆ alkyl-(C═O)NR^(a)R^(b),b) C₀-C₆ alkyl-SO_(m)R^(d), c) C₀-C₆ alkyl-CO₂R^(c), d) C₁-C₆alkyl-OR^(c), e) C₁-C₆ alkyl-NR^(a)R^(b), and f) C₀-C₆ alkyl-(C═O)—C₀-C₆alkyl-OR^(c); R², R³ and R⁵ are hydrogen; R⁴ is selected from: 1) C₆₋₁₀aryl, 2) C₅₋₁₀ heterocyclyl, provided that R⁴ is not pyrididonyl, saidaryl and heterocyclyl optionally substituted with at least onesubstituent selected from R^(b); R^(a) and R^(b) independently areindependently selected from: 1) H, 2) C₁-C₆ alkyl, 3) C₂-C₆ alkenyl, 4)C₂-C₆ alkynyl, 5) C₃-C₁₀ cycloalkyl, 6) C₆-₁₀ aryl, 7) C₅-₁₀heterocyclyl, 9) C₀-C₆ alkyl-SOmR^(d), 10) C₀-C₆ alkyl-CO₂R^(c), 11)C₀-C₆ alkyl-OR^(c), 13) C₀-C₆ alkyl-(C═O)-C₀-C₆ alkyl-OR^(c), saidalkyl, aryl and heterocyclyl are optionally substituted with at leastone substituent selected from R^(d), R^(c) independently is: 1) H, 2)unsubstituted or substituted C₁-C₆ alkyl, 3) unsubstituted orsubstituted C₂-C₆ alkenyl, 4) unsubstituted or substituted C₂-C₆alkynyl, 5) unsubstituted or substituted C₃-C₁₀ cycloalkyl, 6)unsubstituted or substituted C₆-₁₀ aryl, or 7) unsubstituted orsubstituted C₅-₁₀ heterocyclyl; said substitution being at least onesubstituent selected from R^(b); R^(d) independently is: 1)unsubstituted or substituted C₁-C₆ alkyl, 2) unsubstituted orsubstituted C₂-C₆ alkenyl, 3) unsubstituted or substituted C₂-C₆alkynyl, 4) unsubstituted or substituted C₃-C₁₀ cycloalkyl, 5)unsubstituted or substituted C₆-₁₀ aryl, or 6) unsubstituted orsubstituted C₅-₁₀ heterocyclyl; said substitution being at least onesubstituent selected from R^(b); or a pharmaceutically acceptable saltor stereoisomer thereof.
 2. The compound of claim 1, wherein R⁴ is aryl,which is optionally substituted with at least one substituent selectedfrom R^(b); or a pharmaceutically acceptable salt or stereoisomerthereof.
 3. A compound selected from:6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid ethylester; 6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid;6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid amide;6-(4-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carbonitrile; ethyl 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]nicotinate;5-[6-(4-methoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl]-N-methylnicotinamide;N-ethyl-5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]nicotinamide;N-cyclopropyl-5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]nicotinamide;5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-N-propylnicotinamide;5-[6-(3-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-N-methylnicotinamide;N-ethyl-5-[6-(3-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]nicotinamide;5-[6-(3-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-N-propylnicotinamide;N-cyclopropyl-5-(6-pyridin-4-ylpyrazolo[1,5-a]pyrimidin-3-yl)nicotinamide;N-propyl-5-(6-pyridin-4-ylpyrazolo[1,5-a]pyrimidin-3-yl)nicotinamide; ora pharmaceutically acceptable salt or stereoisomer thereof.
 4. Thecompound according to claim 3 selected from:5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-N-methylnicotinamide

or a pharmaceutically acceptable salt or stereoisomer thereof.
 5. Thecompound according to claim 3 selected from:N-ethyl-5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]nicotinamide

or a pharmaceutically acceptable salt or stereoisomer thereof.
 6. Thecompound according to claim 3 selected from:N-cyclopropyl-5-(6-pyridin-4-ylpyrazolo[1,5-a]pyrimidin-3-yl)nicotinamide

or a pharmaceutically acceptable salt or stereoisomer thereof.
 7. Apharmaceutical composition which is comprised of a compound inaccordance with claim 1 and a pharmaceutically acceptable carrier.